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  • Thirty five training subjects were recruited consecutively between May 2010 and August 2011 and 35 control subjects between May 2009 and May 2010 at the Division of Neurodegenerative Diseases, Department of Neurology at Dresden University of Technology. The control subjects participated in a longitudinal olfactory study. Eligible subjects were 18 years of age or older, had received the diagnoses of PD according to UK Brain Bank criteria [15], and were on stable anti-parkinsonian medication for at least 4 weeks prior to study enrollment and during the study. The following exclusion criteria had been defined: Identifiable cause of parkinsonism or signs for atypical parkinsonian disorders, dementia, and psychiatric conditions interfering with study participation. Detailed information about the experiment was given to all participants and written consent was obtained. All aspects of the study were performed in accordance with the Declaration of Helsinki. The study protocol was approved by the local Ethics Board of the Faculty of Medicine of Dresden University of Technology. The training group performed olfactory training over a period of 12 weeks. Patients exposed themselves twice daily to four odors (phenyl ethyl alcohol (PEA): rose, eucalyptol: eucalyptus, citronellal: lemon, and eugenol: cloves). These four odorants were chosen to be representative of four odor categories claimed by Henning [16] in his work on the “odor prism” (Geruchsprisma), where he tried to identify primary odors (compare [17]). These categories are flowery: blumig (e.g., rose), foul: faulig, fruity: fruchtig (e.g., lemon), aromatic: würzig (e.g., cloves), burnt: brenzlig, and resinous: harzig (e.g., eucalyptus). Training patients received four brown glass jars (total volume 50 mL) with one of the four odors in each (1 mL each, soaked in cotton pads to prevent spilling). All jars were labeled with the odor name. Patients were asked to sniff the odors in the morning and in the evening for approximately 10 seconds each. To focus their attention on the training, they were asked to keep a diary in which they rated their overall olfactory abilities each Sunday (data not analyzed). Further, patients received a phone call by one of the experimenters 4 weeks after the training started (1) to ask about the patients' olfactory function and (2) to maintain compliance with the training procedure. Patients in the non-training group were advised to wait and see how the olfactory function would change. Olfactory testing was performed before and after the training period of 12 weeks using the “Sniffin' Sticks” test kit [18] which involves tests for odor threshold, odor discrimination, and odor identification. Using commercially available felt-tip pens, the odorants were presented approximately 2 cm in front of both nostrils for 2 seconds. PEA odor threshold was assessed by a single-staircase, 3-alternative forced choice (3-AFC) procedure. Three pens were presented to the patient in a randomized order, two contained odorless solvent (propylene glycol) and the other an odorant in a certain dilution. The patient's task was to indicate the pen with the odorant. Concentration was increased if one of the blanks was chosen and decreased if the correct pen was identified twice in a row. The mean of the last 4 of a total of 7 reversal points was used as detection threshold (ranging from 1 to 16). A total of 16 odor concentrations were tested starting from a 4% stock solution (dilution ratio 1∶2; solvent propylene glycol). The second subtest assessed the ability of the patient to discriminate different odors. Again, 16 triplets of pens were offered, each including two identical odors and a different one. The patient's task was to indicate the pen which had a different smell. The score was the sum of correct responses ranging from 0 to 16. Both threshold and discrimination testing was performed with the patient being blindfolded. For testing of odor identification, 16 pens containing common odors were offered. The patient had to identify each of the odorants from a list of four descriptors. The sum of the scores from the three subtests resulted in the TDI-score (Threshold, Discrimination, Identification) with a maximum of 48 points. A score of 30.5 points or more indicates normosmia, a score between 16.5 and 30 points indicates reduced olfactory function in terms of hyposmia, and a score of less than 16.5 points indicates functional anosmia. While thresholds for PEA were measured using the single-staircase paradigm within the Sniffin' Sticks test kit (see previously discussed data), thresholds for the other odorants used for training (eucalyptus, eugenol, and citronellal) were assessed by means of the method of ascending limits [19], using a 3-AFC procedure. This procedure was chosen because it is slightly faster than the staircase procedure, although it may be somewhat less reliable [20]. Odors were presented in brown glass jars, similar to the presentation of PEA using the “Sniffin' Sticks”. Two of the jars contained odorless solvent (propylene glycol), the other an odorant in a certain concentration. The patient's task was to indicate the jar with the odorant. Correct identification was assumed when the patient correctly identified the same odor concentration three times in a row. A total of eight odor concentrations for each odor were tested starting from 4% stock solutions (dilution ratio 1∶4; solvent propylene glycol). Between tests of the odorants, subjects rested for approximately 5 minutes to minimize adaptation. Data were analyzed by means of SPSS 19.0 (SPSS Inc., Chicago, Ill, USA). If not mentioned otherwise, all data are displayed as means±standard deviation (SD) or numbers (%), significance level was set at p<0.05 (two-tailed test). Bonferroni tests were used for post-hoc analyses. Pearson statistics were used for correlational analyses.
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